Project Summary Influenza A viruses (IAVs) have caused four pandemics since the turn of the 20th century. The 1918-1919 “Spanish Flu” was the most severe and is estimated to have caused more than 50 million deaths worldwide. A rapid response in the face of the next pandemic depends on the availability of pre-positioned medical countermeasures, including vaccines and therapeutics. These medical interventions are in a constant arms race with rapidly evolving IAV strains that use multiple strategies to escape them. The segmented genome of influenza viruses can reassort when two IAV strains infect the same cell, allowing generation of novel strains in a process called antigenic shift. These new strains, most often arising in an avian reservoir, have been to blame for each of the previous pandemics as humans may have no preexisting immunity. Additionally, the viral error-prone polymerase drives antigenic drift, allowing rapid accumulation of mutations in response to selective pressure to effectively escape the host immune response or antiviral medications. Antigenic drift has driven the emergence of multiple strains that are resistant to each of the six FDA-approved antivirals and underscores the need for next-generation therapeutics. Immunotherapeutics are effective antiviral countermeasures and can be carefully designed with flu evasion strategies in mind. First, epitopes that are highly conserved among the many different IAV strains circulating in multiple hosts are less likely to tolerate resistance mutations, making them an appropriate target for antiviral molecules. Second, bispecific molecules that bind to two distinct epitopes can be employed to decrease the likelihood of complete escape, and in some cases can function synergistically. With these ideal properties of a next-generation IAV immunotherapeutic in mind, we propose to develop a novel bispecific molecule targeting the highly conserved hemagglutinin (HA) stalk and the matrix 2 ectodomain (M2e). Our approach leverages a new class of immunotherapeutics—the ODIN (Orthogonal Dual-Interacting Nanotherapeutic) platform—that combines two natural immune sequence repertoires into single-domain bispecific molecule. ODIN molecules seamlessly merge camelid VHHs with the ultra-long CDR3s (UL-CDR3s) found in a subset of bovine immunoglobulin heavy chains to create a “small with a long reach” bispecific. The small size allows ODIN molecules to access epitopes like the HA stalk and M2e that IAV shields from immune surveillance with glycosylation and spatial localization near the viral membrane. ODINs can also be tailored to provide different mechanisms of action. To generate IAV ODIN molecules, we will immunize cattle with a combination of divergent IAV strains and recombinant proteins to elicit broad-spectrum UL-CDR3s. The lead UL- CDR3s will be combined with complimentary VHHs, yielding a subset of ODIN candidates that will be evaluated for IAV neutralization breadth and potency and the ability to r...